Human monoclonal antibodies have proven to be invaluable in therapeutic applications, either as IgG of conventional size, single chains or domain modules1, 2. Despite the successes there are still major shortcomings in their production, which relies either on specificity selection of available human material and subsequent modification of individual products, or the immunization of the limited availability of transgenic animals, mainly mice3. Target antigen restrictions are widely in place for the use of transgenic mice, as well as large transgenic animals such as cattle, and the development of new specificities is company controlled4-7.
DNA rearrangement and expression of human immunoglobulin (Ig) genes in transgenic mice was pioneered over 20 years ago by stably inserting heavy-chain genes in germline configuration8. Although human antibody repertoires were obtained in these early animals, major improvements, resulting in higher expression levels and exclusive production of human Ig, combined two new strategies: gene knock-out in embryonic stem (ES) cells9 and locus extension on artificial chromosomes10.
Silencing of the endogenous Ig genes by gene targeting in ES cells produced several inactive mouse lines without the ability to rearrange their IgH and Igκ locus or without producing fully functional IgH, Igκ or Igλ, products (summarized in3). More recently zinc finger nucleases (ZFNs) were designed to generate site-specific double-strand breaks in Ig genes, which allowed gene disruption by deletion and non-homologous DNA repair. Injection of ZFN plasmids into fertilized eggs produced Ig silenced rats and rabbits with IgH and IgL disruptions11-13.
Efficient expression of antibodies requires functional regulatory elements in various locations in immunoglobulin loci. Enhancer sequences have been identified near many active genes by nuclease digest and hypersensitivity to degradation. Hypersensitive sites may precede promoter sequences and the strength of their activity was correlated with the DNA sequence. Linkage to reporter genes showed elevated transcription if enhancer function was present (Mundt et al., J. Immunol., 166, 3315[2001]. In the IgH locus two important transcription or expression regulators have been identified, Eμ and the 3′E at the end of the locus (Pettersson et al., Nature, 344, 165 [1990]). In the mouse the removal of the entire 3′ regulatory region (containing hs3a, hs1,2, hs3b and hs4) allows normal early B-cell development but abrogates class-switch recombination (Vincent-Fabert et al., Blood, 116, 1895 [2010]) and possibly prevents the optimization of somatic hypermutation (Pruzina et al., Protein Engineering, Design and Selection, 1, [2011]).
The regulatory function to achieve optimal isotype expression is particularly desirable when transgenic human IgH genes are being used. However, in a number of laboratories, transgene constructs with an incomplete 3′E region, typically providing only the hs1,2 element, led to disappointing expression levels in transgenic mice even when the endogenous IgH locus was knocked-out. This may be one reason why the generation of antigen-specific fully human IgGs from genetically engineered mice has been inefficient thus far. (Lonberg et al., Nature 368, 856 [1994]; Nicholson et al., J. Immunol., 163, 6898 [1999]; Davis et al., Cancer Metastasis Rev. 18, 421 [1999]; Pruzina et al., Protein Engineering, Design and Selection, 1,[2011].
In the rat, the 3′E region has only been poorly analyzed. A comparison of mouse and rat sequences does not allow identification of hs4, the crucial 4th E element with additional important regulatory sequences further downstream (Chatterjee et al., J. Biol. Chem., 286,29303 [2011]). This could mean the region is not present in the rat, and perhaps not as important as in the mouse, or it could be absent in the analyzed rat genome sequences.
Still needed are methods and materials for the optimal production of immunoglobulins or antibodies having human idiotypes using transgenic animals, which are useful for treating humans in a broad range of disease areas.